Plant Physiol. (1990) 94, 1721-17270032-0889/90/94/1721/07/$01 .00/0

Received for publication May 8, 1990Accepted August 7, 1990

Tracheid Production in Response to Changes in the InternalLevel of Indole-3-Acetic Acid in 1-Year-Old

Shoots of Scots Pine1

Bjorn Sundberg* and C. H. Anthony LittleDepartment of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, S-901 83 Umea,Sweden (B.S.), and Forestry Canada-Maritimes Region, Fredericton, New Brunswick, E3B 5P7, Canada (C.H.A.L.)

ABSTRACT

Different concentrations of indole-3-acetic acid (IAA) were ap-plied in lanolin to 1-year-old shoots of Pinus sylvestris (L.) in amanner known to stimulate cambial activity. The intemal concen-tration of free IAA was measured at a distance below the appli-cation point by combined gas chromatography-selected ion mon-itoring-mass spectrometry using [13CJ]IAA as a quantitative inter-nal standard, and related to the production of tracheids at thesame site. The experiment was performed with: (a) debuddedcuttings, where the major source of endogenous IAA, the apicalbuds, were replaced with exogenous IAA, and (b) intact, attachedshoots, where endogenous IAA was supplemented by applyingIAA around the circumference of the shoot. In both experimentalsystems, an increase in the intemal IAA level was positivelyrelated to increased tracheid production. It was also demon-strated that the concentration of intemal IAA measured at thesampling site was comparable with endogenous IAA levels foundin intact control shoots, and that a wide range of applied IAAconcentrations was associated with a relatively small range ofintemal IAA levels.

One-year-old conifer shoots are often used when investigat-ing the role of IAA in the regulation of cambial activity, asreflected in wood formation. Numerous studies involving theapplication of ['2C]- and ["4C]IAA to this experimental systemhave demonstrated that IAA is transported in a basipetallypolar fashion and can substitute for the decapitated elongatingbud in maintaining tracheid production along the entire shootaxis. The concentration ofexogenous IAA is positively relatedto subsequent tracheid production (for recent reviews, seerefs. 4 and 8). It has also been observed that defoliating thecurrent-year shoot decreases both the production of tracheidsand the level of endogenous IAA in the subjacent 1-year-oldinternode (13). Considered together, these observations sug-gest that young leaves are the primary site of IAA synthesisand that basipetally transported IAA plays a major role in themechanism regulating the rate of tracheid production in the1-year-old shoot. This interpretation is based on results ob-tained with an experimental system in which the level ofendogenous IAA inevitably becomes limiting for tracheid

'Supported by the Swedish Council for Forestry and AgriculturalResearch.

production. Consequently, treatment with exogenous IAAwould be expected to induce a response. However, it has yetto be demonstrated that tracheid production in an intact,vigorously growing shoot can be changed by altering theinternal IAA level.The relevance of results obtained with exogenous IAA for

elucidating the role(s) of endogenous IAA in the control ofprocesses such as tracheid production has been criticized fortwo main reasons (15, 16). First, the concentrations of exog-enous IAA used in dose-response experiments are muchhigher than the levels of endogenous IAA normally found inplant tissues. Second, the range in the concentration ofappliedIAA greatly exceeds the variation in endogenous IAA level.This criticism assumes that the internal IAA concentrationattained after IAA treatment is similar to the concentrationof the exogenous IAA. However, such may not be the case,because the internal level also depends on how much of theapplied IAA is absorbed, transported, and metabolized.

In the present study, we investigated how different concen-trations of applied IAA affected the internal concentration offree IAA and the production of tracheids in 1-year-old Pinussylvestris (L.) shoots at a distance below the application point.This approach has allowed us to relate tracheid production tothe amount of free IAA that was actually present at the siteof action in response to the treatment with exogenous IAA.Two dose-response experiments were performed. The firstinvolved the classic system for investigating cambial activity,in which case IAA was applied to the apical end ofdebudded,1-year-old cuttings that were dormant when the experimentbegan. In the second experiment, IAA was applied aroundthe circumference of budded 1-year-old shoots left attachedto field-grown trees, the goal being to elevate the endogenouslevel of free IAA in intact shoots that were already producingtracheids at the start of the experiment.

MATERIALS AND METHODS

Plant Material and IAA Application

Experiment 1

On March 15, 1988, when the cambium was in the dor-mancy stage of quiescence, seven cuttings were collected fromeach of 50 Scots pine (Pinus sylvestris L.) trees, 6 to 8 m tall,located in a spaced, naturally regenerated stand 5 km southof Umea, Sweden (63°50' north, 20°20' east). The cuttings

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Plant Physiol. Vol. 94, 1990

were obtained by clipping the current-year terminal shootfrom the main branches (secondary axes) positioned in thetwo to four uppermost whorls on the stem (primary axis). Aneighth cutting was similarly collected from 10 of the same 50trees. In the laboratory, the latter group of 10 cuttings washarvested, as described below, for measurement of the endog-enous IAA level at the time of collection. The remaining 350cuttings (seven from each of 50 trees) were trimmed undertap water at the proximal end to a length of 22 cm, startingat the base of the apical overwintering buds. These buds wereremoved from six of the seven cuttings per tree, and onedebudded cutting per tree was treated on the apical cut surfacewith 0, 0.1 1, 0.33, 1, 3, or 9 mg IAA g-' lanolin (0.8 g lanolin/cutting) after defoliating the distal 1 cm of length. The seventhcutting per tree was left with the apical buds intact to serve asa "budded control." The cuttings were placed upright inbeakers with their base immersed in glass-distilled water andcultured in a controlled environment chamber having a pho-ton flux density of 240 jumol s-' m-2 from Osram HQI-TS400 W/DH metal halogen lamps, a temperature of 240/1 3°C(day/night), a photoperiod of 18 h, and a relative humidityof about 75%. At weekly intervals for 35 d, the cuttings of 10randomly selected trees were harvested, while the water andthe IAA-lanolin applied to all remaining cuttings were re-placed after removing a 1-mm slice from either end. At thetime of harvesting, two sections, one 8 cm long and the other1 cm long, were removed from each cutting 2 to 10 and 10to 11 cm below the apex, respectively. The long section wasimmersed in liquid N2, then stored at -80°C until the basal 1cm of it (i.e. 9-10 cm below the apex) was used to measureIAA. Tracheid production was determined in the short sec-tion, at the end located immediately below the 1-cm pieceused for IAA measurement. In previous work with this exper-imental system, we observed that apically applied IAA inducesthe formation of compression wood, and of callus within thecortex, below the application point for approximately 1 cm,but not at a distance of about 10 cm (9).

Experiment 2

Forty Scots pine trees, 2 to 3 m tall and bearing at leastfive, 20-cm-long branches in the uppermost whorl on themain stem, were selected in a spaced, naturally regeneratedstand located at Svartberget Experimental Forest, Vindeln, 60km northwest of Umea, Sweden (64°14' north, 19°46' east).On June 16, 1988, shortly after the start of tracheid produc-tion and bud expansion, one shoot per tree was treated with0, 0.33, 1, or 3 mg IAA g-' lanolin. This was applied aroundthe entire circumference (i.e. laterally) along 1.5 cm of theshoot axis, starting 5 cm below the apical elongating buds.The epidermis of the treated region was carefully removedwith a scalpel immediately before application. After applica-tion, the treated region was covered with aluminum foil. Thefifth shoot per tree was left totally undisturbed to serve as theintact (untreated) control. At weekly intervals for 35 d, theshoots of eight randomly selected trees were harvested. At thesame time, the IAA-lanolin applied to the shoots of theremaining trees was replaced with fresh material, after wipingaway the old with a tissue. The lengths of the current-yearterminal shoot and subjacent 1-year-old shoot (i.e. internode)

were also measured in all unharvested experimental branchesafter 21 d of treatment. At harvest, the 1-year-old internodewas defoliated, and a 5-cm-long section was removed 2 to 7cm below the application point. This section was immersedin liquid N2, then stored at -80°C until used to measure IAA.The remainder of the 1-year-old internode was stored at-20°C until used to determine tracheid production.

Measurement of Tracheid Production

With one exception described below, tracheid productionwas measured in transverse, nonembedded sections that werehandcut at one or more locations along the shoot axis. Theexact position(s) are detailed in "Results." The sections werestained in a saturated aqueous solution of phloroglucinol in20% HCI, which reacts with lignin, and mounted in Canadabalsam. Tracheid production was measured as number ofnewtracheids per radial file and/or radial extent of new xylem.These measurements were recorded at eight equidistant pointsaround the circumference, starting from the last-formed trach-eid in the latewood of the previous year's annual ring (6). Themeasurement included only those new tracheids reacting withthe stain, hence in which at least some secondary wall ligni-fication had occurred. Hereafter, these are referred to aslignified tracheids. In addition, the radial widths of the bark(i.e. all tissues located outside the cambium) and of theprevious year's xylem were measured. In experiment 2, trach-eid production was expressed as the ratio of current-yearxylem width to previous-year xylem width, while bark growthwas expressed as the ratio of total bark width to previous-yearxylem width, to obviate initial differences in shoot size. Trach-eid diameter was estimated either by dividing xylem radialwidth by tracheid number (experiment 1) or by measuringthe radial extent of the 10 most recently formed tracheids(experiment 2).The method of measuring tracheid production described

above is rapid and simple to perform, but has the disadvantagethat tracheids with little or no secondary wall developmentare excluded from the measurement because they do not reactwith the indicator stain and, in any event, are crushed duringthe sectioning process (6). To record tracheids in early stagesof differentiation, therefore, representative samples were em-bedded in paraffin after fixation in formalin-acetic acid-ethanol (1). These samples comprised one-quarter of thecircumference and were obtained from the midpoint of ex-periment 1 cuttings that either bore buds (budded control) orhad been debudded and treated apically with 0, 0.1 1, or 1 mgIAA g-' lanolin. Transverse sections were cut 8 to 10 Amthick on a sledge microtome, stained in hematoxylin-safranin(1), and mounted in Canada balsam. The number oftracheidsthat were expanding radially, whose secondary wall was thick-ening, and that had completed differentiation was counted infive radial files per cutting. Radially enlarging tracheids weredistinguished from tracheids undergoing secondary wall de-velopment by their having a relatively narrow radial diameter.The thickening of the secondary cell wall was assumed to befinished when the width of the tangential wall visibly equaledthat in old tracheids in the same file.

1 722 SUNDBERG AND LITTLE

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TRACHEID PRODUCTION IN RELATION TO THE INTERNAL IAA LEVEL IN PINE

Measurement of IAA

IAA was measured using GC-selected ion monitoring-MSwith ['3C6]IAA as a quantitative internal standard, as de-scribed in detail elsewhere (12). In brief, the sample wasobtained by peeling the bark and scraping the exposed surfaceon the xylem side with a scalpel. The bark peeling andscrapings for all shoots per treatment and harvest date werepooled and ground in liquid N2. Two 0.5-g aliquots wereextracted for 1 h in 5 ml of 0.05 M Na-phosphate buffer, pH7.0, containing 0.02% of the antioxidant sodium diethyldi-thiocarbamate with ['3C6]IAA (Cambridge Isotope Laborato-ries) added as internal standard. The extract was purified byneutral-acidic diethyl ether partitioning, and the acidic etherportion was methylated, then subjected to reversed-phase C18HPLC. The HPLC mobile phase consisted of 50% methanolin 1% acetic acid and was delivered at a flow rate of 1 mlmin-' by Waters Model 501 pumps. The sample was intro-duced by a Waters 712 WISP onto a 10 cm x 8 mm i.d. 4-,um Nova-Pak C18 cartridge fitted in a RCM 8 x 10 module(Waters Associates). The IAA-methyl ester fraction was col-lected, silylated, and analyzed by GC-selected ion monitoring-MS. Chromatography was done on a 25 m x 0.25 mm i.d.SE-30 column with a film thickness of 0.25 um (QuadrexCompany) using He as a carrier gas at a flow rate of 1 mlmin-'. The GC-MS was a Hewlett-Packard 5890 gas chro-matograph linked via a direct inlet to a 5770 Hewlett-Packardmass selective detector. The ratio of m/z 202:208 was used tocalculate the endogenous content of IAA by the isotopedilution equation as modified by Cohen et al. (2), and theratios of m/z 202:261 and 208:267 were used to check forinterference.

RESULTS

Experiment 1: Effect of Apically Applied IAA on theProduction of Tracheids and the Internal Concentrationof IAA in Debudded Cuttings

The terminal bud on the budded control cuttings elongatedalmost threefold during the culture period. Tracheids withlignified secondary walls were present in this material on day14, and their production continued at a generally constantrate throughout the experiment (Fig. l A). In contrast, cambialreactivation was almost totally inhibited in debudded cuttingstreated with plain lanolin. Applying IAA to the apical cutsurface of debudded cuttings promoted cambial activity at allconcentrations used. After 21 d in culture, tracheid produc-tion increased progressively with increasing IAA concentra-tion, up to 3 mg g-' lanolin. This relationship was apparenton day 21 for all types of tracheids, viz. lignified tracheids(Fig. lA), tracheids undergoing radial expansion and second-ary wall thickening (Fig. 2, A and B), fully differentiatedtracheids (Fig. 2C), and tracheids in all stages ofdifferentiationcombined (Fig. 2D). Raising the IAA concentration from 3to 9 mg g-' lanolin, however, resulted in fewer tracheids (Fig.IA). After 21 and 28 d of treatment, the rate of tracheidproduction decreased in debudded cuttings receiving 3 and 1

mg IAA g-' lanolin, respectively (Figs. IA and 2D). In con-

trast, after day 21, the tracheid production rate increased indebudded cuttings treated with 0.11 and 0.33 mg IAA g-'

10

ul

z

IDIu-

8

6

4

2

0l

150_

0)

0)C

100o-

50-

01

0 10 20

DAY S

30 40

Figure 1. Lignified tracheid number (A) and internal IAA concentration(B) at the midpoint of 1 -year-old cuttings that either bore buds(budded control; U) or were debudded and treated apically with 0(A), 0.11 (A), 0.33 (E1), 1 (0), 3 (0), or 9 (x) mg IAA g-' lanolin for upto 35 d. Vertical line indicates (A) SE, n = 10, and (B) the range of theduplicate measurements of 10 pooled shoots, when larger than thesymbol. In A, for each of the sampling days 21, 28, and 35, everymean marked with a * is significantly different (P - 0.05, paired t test)from the next subjacent one; for day 14, 1 > 0.33, 3 and 9 > 0.11and budded control > 0.

lanolin (Figs. IA and 2D). Thus, the number of tracheidsproduced at the end of the culture period did not differ indebudded cuttings subjected to 0.11, 0.33, 1, and 3 mg IAAg-' lanolin. The rate of tracheid production in debuddedcuttings treated with 9 mg IAA g-' lanolin remained constantthroughout the experimental period. The cells at the apicalcut surface of these cuttings had a necrotic appearance eachtime that the IAA-lanolin mixture was replaced.The average radial diameter of all lignified tracheids did

not differ significantly between treatments or culture periods(data not shown).

A 1

.333-

.11

9

0

B

l

1 723

A

.I

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Plant Physiol. Vol. 94, 1990

1 *4 5 1 *(J

0

control; *) or weeeudeadtraedaiclyi control5 ~

0 10 20 -30 0 10 20 30 40

DAYS

Figure 2. Number of radially expanding tracheids (A), tracheids form-

ing their secondary wall (B), fully differentiated tracheids (C), anddifferentiating plus fully differentiated tracheids, ie. A + B + C (D) atthe midpoint of 1-year-old cuttings that either bore buds (buddedcontrol; x) or were debudded and treated apically with 0 (U), 0.11

(0), or 1 ()mg IAAg11 lanolin for up to 35 d. Vertical line indicates

SE, = 10. For each sampling day within each tracheid category,

every mean marked with a * is significantly different (P t0.05, pairedtest) from the next subjacent one, except for day 14 in A, where 1

> 0.1 1 and budded control > 0.

The internal IAA concentration in all materials changed

markedly during the experimental period (Fig. eB). In debud-

ded cuttings treated with plain lanolin, the IAA level declinedduring the first 14 d, and subsequently did not vary. A

different pattern was observed in budded control cuttings andin debudded cuttings treated with exogenous IAA. In these,

the intemal IAA level peaked on day 7, decreased through

days 14 to 21, and thereafter remained relatively constant.

On days 7 and 14, the internal IAA level increased progres-sively with increasing concentration of applied IAA, up to 3

mg g' lanolin. The approximately 30-fold range between the0.11 and 3 mg g' lanolin concentrations of applied IAAinduced only an approximately 2.5-fold range in internal IAA

level. Treatment with 9 mg IAA g'1 lanolin resulted in an

interal IAA concentration between the levels found in de-

budded cuttings treated with and 0.33 mg IAA g' lanolin.The internal IAA level in budded control cuttings was most

comparable with that measured in debudded cuttings treated

with 0.33 mg IAA g'1 lanolin. On days 21, 28, and 35, a

similar internal IAA level was observed in all cuttings except

those treated with 0 mg IAA g-' lanolin.

Two lines of evidence indicate that the differences in inter-

nal IAA concentration induced by exogenous IAA treatment

on days 7 and 14 were real. First, the hypothesis that all

applied IAA concentrations had the same effect on the inter-

nal IAA content was rejected (P = 0.007), using log-trans-

formed data in a two-way analysis of variance, the modelbeing:

log r,i = ai + bj + eij

where r is the response (internal IAA level), a is the effect oftreatment, b is the effect of time and different plant material,eij is the error, i is 1, . . . 5 (treatment), and j is 1, 2 (time).Second, the probability of exogenous IAA concentrationsinducing the same order of internal IAA levels on two succes-sive occasions by chance was only 1/ 120.

Experiment 2: Effect of IAA Applied around theCircumference on Radial Growth and Intemal IAAConcentration in Budded, Attached Shoots

All concentrations of exogenous IAA visibly promotedradial growth at the pont of application. The anatomy of thispromotion was investigated in shoots treated with 0 and 1 mgIAA g-' lanolin for 4 weeks. Massive callus formation in thecortex increased the radial width of the bark between about 1and 3 cm above and below the application point, respectively(Fig. 3A). Xylem radial width was also markedly increased inthe vicinity of the application point. Many of the tracheidsproduced in this region had characteristics of compressionwood tracheids (14), viz. round shape, thick secondary wall,and, judging from the intensity ofthe staining with phloroglu-cinol-HCl, high lignin content. However, in contrast to thelocalized bark swelling, an increase in xylem radial width wasevident for at least 7 cm below the IAA application point. Atthis distance, there was no compression wood formation, andthe IAA-induced promotion of xylem radial width was dueentirely to an increase in tracheid number (Fig. 3B). Tracheiddiameter was promoted only at and immediately below theapplication point (Fig. 3C), where compression wood trach-eids were present. Because we wanted to determine the rela-tionship between internal IAA level and tracheid productionwhere radial growth was normal and earlywood-type tracheidswere being produced, we used the piece of shoot located 6 to7 cm below the application point for the IAA determinationand measured tracheid production immediately below thispiece.Throughout the experimental period, tracheid production

in shoots treated with plain lanolin was not different thanthat in intact control shoots (Fig. 4A). Thus, the method usedto apply IAA did not significantly affect cambial activity. Allconcentrations of exogenous IAA promoted tracheid produc-tion to the same extent. The difference in xylem radial widthbetween the IAA-treated shoots and the control shoots wasestablished during the first 14 d of treatment. Thereafter, thecambial growth rate was similar in all shoots.

Applying IAA around the circumference of the 1-year-oldshoots resulted in longitudinal growth being inhibited in thecurrent-year shoots borne on the distal end of the treatedshoot. The degree of this inhibition increased with increasingIAA concentration (Table I). The highest concentration ofexogenous IAA also caused the current-year shoots in sometrees to curl abnormally.The internal IAA concentration was the same in the shoots

treated with plain lanolin as in intact control shoots, andchanged little during the experimental period (Fig. 4B). In

1 724 SUNDBERG AND LITTLE

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TRACHEID PRODUCTION IN RELATION TO THE INTERNAL IAA LEVEL IN PINE

II-a

NCD-j

N

I

3

z0

I-

I.-

E

0

LUI-

CIO

I uM:

CZI.

. ,I.

-&-- ,* * 0-

<4 2 0 0 2 4 6 <

CENT METERS

Figure 3. Radial width of the bark and of the 1988 lignified xylem(A), lignified tracheid number (B), and lignified tracheid diameter (C)along the length of 1-year-old attached shoots treated around theircircumference with 0 (0) and 1 (x) mg IAA g-' lanolin for 28 d. Thickarrow indicates where exogenous IAA was applied. Thin arrow indi-cates where internal IAA was measured. Vertical line indicates SE, n= 8. * Significant difference between the means for each measure-ment point (P < 0.05, paired t test).

contrast, treatment with exogenous IAA had obviously ele-vated the internal IAA concentration by day 7. Despite a 10-fold difference in the concentration of applied IAA, the re-sulting levels of internal IAA were similar. The internal IAAconcentration was also elevated on day 14, whereas by day21 it was similar in all shoots.

DISCUSSION

Experiment I clearly demonstrates that applying IAA to theapex ofdebudded cuttings results in an internal concentrationof free IAA that is physiologically relevant, i.e. similar to thatfound in cuttings with flushing buds. However, the relation-ship between the internal IAA level and tracheid productionin this experimental system is not readily assessed, for tworeasons. First, there was a delay between the start of the

x

\

Iam

Ir-

CDCD

0.7 F0.33,1,3A

0.6 -0

Intactcontrol0.5 -

0.4F

0.3 p

0.2

400B

0.33

3

300_

200 FIntactcontrol

0

loo1-

[

0 10 20

DAY S

30 40

Figure 4. Radial width of lignified xylem (A) and internal IAA concen-tration (B), measured 6 to 7 cm below the application point of 1-year-old attached shoots that were either left intact (intact control; U) ortreated around their circumference with 0 (E, 0.33 (0), 1 (0), or 3(x) mg IAA g-1 lanolin for up to 35 d. Vertical line indicates (A) SE, n= 8, and (B) the range of the duplicate measurements of eight pooledshoots, when larger than the symbol. In A, * significant difference (P- 0.05, paired t test) between the mean for each of the 0.33-, 1-,and 3-mg IAA g-' lanolin treatments and the mean for the 0-mg IAAg-1 lanolin treatment; for all sampling days, there was no significantdifference among the means for the 0.33-, 1-, and 3-mg IAA g-1lanolin treatments, and between the means for the 0-mg IAA g-1lanolin treatment and the intact control.

w

l X |

1 725

F

I I

L

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SUNDBERG AND LITTLE

Table I. Length of the Current-Year Terminal ShootShoot length was measured after 21 d of treatment with exoge-

nous IAA and is expressed as the ratio of terminal shoot length tolength of the subjacent 1-year-old internode, to obviate initial differ-ences in branch size. Means of 24 replicates, ± SE.

IAA g-' Lanolin Ratio

Untreated 0.714 ± 0.0280mg 0.711 ± 0.0280.3 mg 0.658 ± 0.029*1 mg 0.626 ± 0.027*3 mg 0.593 ± 0.027*

* Mean significantly different (P < 0.05), paired t test) when com-pared with the mean of next higher value.

experiment and the occurrence of recordable (i.e. lignified)tracheids, and the internal IAA concentration varied duringthis interval (Fig. 1). Second, tracheid production in thedebudded cuttings treated with 3 and I mg IAA g-' lanolindeclined after 21 and 28 d in culture, respectively (Fig. IA),indicating that factors essential for cambial growth were be-coming deficient, presumably because the experimental sys-tem lacked roots. To facilitate interpretation, therefore, weexamined the data for only the first 21 d of culture, duringwhich period cambial reactivation progressed normally. Onday 21, there was a positive linear relationship between trach-eid number and the level of internal IAA, both measured ondays 7 and 14, as well as with the sum of the IAA levels foundon days 7, 14, and 21 (Fig. 5A). The slope of the regressionwas significant for all three cases (P c 0.05). In contrast,tracheid number was related to the log10 of the concentrationof exogenous IAA, up to 3 mg g-' lanolin (Fig. 5B). It followsfrom Figure 5 that the relatively small number of tracheidsproduced in debudded cuttings treated with 9 mg IAA g-'lanolin was associated with a low concentration of internalfree IAA, rather than a high, inhibitory level. The 9 mg IAAg-' lanolin concentration might thus have resulted in a de-creased internal IAA level because of injury to cells at theapical cut surface, thereby inhibiting the uptake of appliedIAA. Alternatively, high exogenous IAA may have promotedincreased IAA catabolism and/or conjugation.Although the results for the first 21 d of experiment I

indicate that tracheid production is positively related to theinternal IAA concentration, two additional observations dem-onstrate that the amount of tracheid production induced bya particular internal IAA level varies, thereby emphasizingthe importance of other factors. First, tracheid productionwas generally slower in budded control cuttings than in de-budded cuttings treated with 0.11 and 0.33 mg IAA g-'lanolin, whereas the internal IAA level was higher in thebudded control cuttings (Fig. 1). This discrepancy is attributedto nutriment being diverted from the cambial sink to thesinks associated with longitudinal growth in budded controlcuttings. Second, the rate of tracheid production towards theend of the culture period in debudded cuttings treated with0.1 1 and 0.33 mg IAA g-' lanolin was similar to that exhibitednear the beginning ofthe experiment in the debudded cuttingsreceiving and 3 mg IAA g-' lanolin. However, the internal

IAA concentration in the former cuttings was lower duringthe first 21 d, and comparable subsequently (Fig. 1).

It has been hypothesized that a high endogenous level ofIAA increases the rate of tracheid differentiation, resulting inreduced tracheid diameter (1 1). If this hypothesis is correct,then the differences in lignified tracheid number induced byIAA treatment on day 21 (Fig. lA) might be due to variationin the rate of production of tracheids in this stage of differ-entiation only. However, our data show that the.lignifiedtracheid number reflected the number of tracheids in theearlier differentiation stages ofradial expansion and secondarywall development, as well as of tracheids in all differentiationstages combined (Figs. 1A and 2). Hence, we conclude thatthe relationship between internal IAA level and tracheidnumber is unaffected by tracheid differentiation stage. Thedata also suggest that a high level of internal IAA is notassociated with an obvious decrease in tracheid diameter.The initial rise and subsequent decline of internal IAA

concentration observed in debudded shoots treated apicallywith exogenous IAA (Fig. 1B) has also been found in bothcuttings and attached shoots of Picea sitchensis (Bong.) Carr.(7). Although experiments with ['4C]IAA showed that thesystem that transported IAA basipetally in P. sylvestris (9)and Abies balsamea (L.) Mill. (5) cuttings still functionedafter at least 5 weeks in culture, it was also evident that thecapacity of the transport system declined over time (5). Thisdecline would result in the transport system being saturatedby increasingly lower concentrations of applied IAA, whichmight explain why the internal IAA level on days 21, 28, and35 no longer varied with the concentration of exogenous IAA(Fig. lB).

In experiment 2, the promotion of tracheid productioninduced by applying IAA around the circumference of intact,attached shoots was established during the first 14 d of treat-ment, when the internal IAA concentration was increased(Fig. 4). This observation indicates that the level of endoge-nous IAA in intact shoots is not optimal for tracheid produc-tion. Laterally applied IAA has also been observed to increasetracheid production in intact seedlings ofA. balsamea (7) andP. sitchensis (3, 10). In these experiments, however, the inter-nal IAA level was not determined, and tracheid production

I

In

200 250 300 350 400 10-

IAA, ng (g FW)V'

100 101

IAA, mg (g lanolin) 1

Figure 5. Relationship after 21 d between the number of lignifiedtracheids and the sum of the intemal IAA concentrations measuredon days 7, 14, and 21 (A) and the concentration of exogenouslyapplied IAA (B) in 1-year-old cuttings that were debudded and treatedapically with 0.11 (A), 0.33 (1), 1 (0), 3 (@), or 9 (a) mg IAA g-'lanolin.

_ 1 I I . ,1,,.,II II1

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TRACHEID PRODUCTION IN RELATION TO THE INTERNAL IM LEVEL IN PINE

was measured at the application point, or only 1 cm below,where an internal IAA level considerably higher than the one

we measured would be expected.The increase in tracheid production induced by applying

IAA around the circumference extended for a much longerdistance below than above the application point (Fig. 3). Thisindicates that exogenous IAA did enter the basipetally polartransport pathway. However, it probably was also transportedacropetally, as evidenced by the inhibition ofextension growth(Table I) and the occurrence of curling in the current-yearshoots borne on the distal end of the 1-year-old shoots. Inaddition, apically transported exogenous IAA may havecaused a reduction in the synthesis of endogenous IAA. Thiswould explain not only why the internal IAA concentrationin the IAA-treated shoots was similar despite a 10-fold differ-ence in exogenous IAA concentration, but also why theinternal IAA level declined after day 7 (Fig. 4B). However,these findings may also reflect a difference in IAA uptake,catabolism, and/or conjugation.

Considering experiments 1 and 2 together, it is evident for1-year-old pine shoots that treatment with exogenous IAAcan temporarily increase the internal free IAA concentrationat a distance below the application point. However, the levelof internal IAA induced is much smaller than the concentra-tion of IAA applied, and applying a wide range of IAAconcentrations results in a relatively small range in internalIAA levels. It is also apparent that the rate ofcambial activity,as measured by tracheid production, can be increased duringthe early part of the growing period by raising the concentra-tion of internal IAA. However, it remains to be demonstratedthat a variation in tracheid production within and betweenintact, unmanipulated trees is reflected in different endoge-nous IAA levels.

ACKNOWLEDGEMENT

We thank Marie Nygren for technical assistance.

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